1. Field of the Invention
This invention relates to mechanical test systems and more specifically to biological indicators for use in determining the effectiveness of gas or steam sterilization.
2. Description of the Prior Art
Chemical systems wherein components or reactants of the system are kept separate prior to initiating a specified indicating reaction upon mixing of the reactants are widely used, particularly in the health care industry, for various chemical, biochemical, or biological tests. In the latter area, which typically uses microorganisms as a testing medium, a nutrient growth media or component thereof is kept separate from the microorganism until the test is performed.
In fermentation tests, for example, as taught in U.S. Pat. No. 3,791,930, a solid media substrate, inoculated with a test strain of microorganisms, has sugar supported above the media substrate. When the sugar is moved onto the media substrate a characteristic fermentation reaction takes place.
In the case of biological fermentation indicators for use in steam or ethylene oxide sterilization, a nutrient broth is typically hermetically sealed in an encapsulated inner glass ampul and microorganism spores are disposed in the cavity of the surrounding enclosure. In a conventional biological indicator of this type, the spores are inoculated on a disc which is disposed in a transparent closed vial. The vial will have means for admitting the sterilant gas or steam, as for example a semipermeable membrane or an adjustable closure that admits steam or gas during the sterilization cycle. The vial will also contain encapsulated growth media such as sterile trypticase soy broth and glucose, and a pH indicator chemical, such as phenol red. These materials are encapsulated in a frangible ampul within the outer vial. The indicator is placed in a gas or ethylene oxide sterilization chamber along with the goods to be sterilized so that the spores within the indicator's vial are exposed to the sterilization along with, and in the same manner as, the goods being sterilized. Upon expected completion of sterilization the indicator is withdrawn from the chamber for testing. The inner frangible ampul is broken, releasing the media and pH chemical solution to mix with any microorganism spores that may have survived the sterilization. The vial is then incubated for a period of from five (5) to seven (7) days at temperatures of from 35 to 55 degrees Centigrade depending on the strain of spores used. If viable bacterial spores are present, their germination and growth will utilize the carbohydrate, glucose, that is in the growth media. Utilization of carbohydrate in the media produces acidic by-products which lower the pH of the solution causing a visible color change in the pH indicator and thereby in the solution. No color change indicates no bacterial growth and a satisfactory sterilization. When phenol red is used as the pH indicator chemical, its initial red color becomes yellow in acid; yellow indicates the presence of viable bacteria and an unsatisfactory sterilization.
The spore bearing microorganism traditionally used for ethylene oxide sterilization is Bacillus subtilis var. globigii; Bacillus stearothermophilus is used in steam sterilization because of its greater resistance to moist heat.
The basic principle of the biological indicator is thus a fermentation of viable bacteriological spores which utilizes carbohydrate and produces an acid by-product. If, however, the carbohydrate content of the nutrient growth media is depleted by consumption by the microorganisms, the microorganisms will then begin to utilize the peptones in the nutrient broth. The byproducts of such metabolism, are alkaline and thus increase the pH of the broth thereby causing the color to revert to red with the appearance of a negative (no growth--no surviving bacteria) test result. This effect is very likely in indicators for ethylene oxide sterilization using Bacillus subtilis, which is known to be a weak carbohydrate ferment. Thus in formulating the nutrient broth for these indicators, an excess of carbohydrate had to be added to insure an accurate test result. However, when excess carbohydrates was used in the nutrient broth of indicators using Bacillus stearothermophilus in steam sterilization, the high temperatures and long periods of time of steam sterilization broke down the carbohydrate, forming an acid which lowered the pH. The phenol red, sensitive to this decrease, changed to an orange color, giving the appearance of what is termed in the art as a "false positive" (growth). Thus different indicators have been required for steam sterilization and ethylene oxide sterilization.